RESUMO
To date, no study has demonstrated that soluble Fas ligand (sFasL)-mediated inflammation is regulated via interaction with Fas in vivo. We found that FasL interacts specifically with tumor necrosis factor receptor superfamily (TNFRSF)10B, also known as death receptor (DR)5. Autoantibody-induced arthritis (AIA) was attenuated in FasL (Faslgld/gld)- and soluble FasL (FaslΔs/Δs)-deficient mice, but not in Fas (Faslpr/lpr and Fas-/-)- or membrane FasL (FaslΔm/Δm)-deficient mice, suggesting sFasL promotes inflammation by binding to a Fas-independent receptor. Affinity purification mass spectrometry analysis using human (h) fibroblast-like synovial cells (FLSCs) identified DR5 as one of several proteins that could be the elusive Fas-independent FasL receptor. Subsequent cellular and biochemical analyses revealed that DR5 interacted specifically with recombinant FasL-Fc protein, although the strength of this interaction was approximately 60-fold lower than the affinity between TRAIL and DR5. A microarray assay using joint tissues from mice with arthritis implied that the chemokine CX3CL1 may play an important downstream role of the interaction. The interaction enhanced Cx3cl1 transcription and increased sCX3CL1 production in FLSCs, possibly in an NF-κB-dependent manner. Moreover, the sFasL-DR5 interaction-mediated CX3CL1-CX3CR1 axis initiated and amplified inflammation by enhancing inflammatory cell influx and aggravating inflammation via secondary chemokine production. Blockade of FasL or CX3CR1 attenuated AIA. Therefore, the sFasL-DR5 interaction promotes inflammation and is a potential therapeutic target.
Assuntos
Artrite/imunologia , Autoanticorpos/efeitos adversos , Proteína Ligante Fas/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Linhagem Celular , Humanos , Células Jurkat , CamundongosRESUMO
O-linked ß-N-acetylglucosamine (O-GlcNAc) is a post-translational modification which occurs on the hydroxyl group of serine or threonine residues of nucleocytoplasmic proteins. It has been reported that the presence of this single sugar motif regulates various biological events by altering the fate of target proteins, such as their function, localization, and degradation. This study identified SMAD4 as a novel O-GlcNAc-modified protein. SMAD4 is a component of the SMAD transcriptional complex, a major regulator of the signaling pathway for the transforming growth factor-ß (TGF-ß). TGF-ß is a powerful promoter of cancer EMT and metastasis. This study showed that the amount of SMAD4 proteins changes according to cellular O-GlcNAc levels in human lung cancer cells. This observation was made based on the prolonged half-life of SMAD4 proteins. The mechanism behind this interaction was that O-GlcNAc impeded interactions between SMAD4 and GSK-3ß which promote proteasomal degradation of SMAD4. In addition, O-GlcNAc modification on SMAD4 Thr63 was responsible for stabilization. As a result, defects in O-GlcNAcylation on SMAD4 Thr63 attenuated the reporter activity of luciferase, the TGF-ß-responsive SMAD binding element (SBE). This study's findings imply that cellular O-GlcNAc may regulate the TGF-ß/SMAD signaling pathway by stabilizing SMAD4.
Assuntos
Acetilglucosamina/química , Neoplasias da Mama/patologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Neoplasias Pulmonares/patologia , Processamento de Proteína Pós-Traducional , Proteólise , Proteína Smad4/química , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Feminino , Glicogênio Sintase Quinase 3 beta/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Serina , Transdução de Sinais , Proteína Smad4/genética , Proteína Smad4/metabolismo , Treonina , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Células Tumorais Cultivadas , Ubiquitina/metabolismoRESUMO
The Hippo pathway controls organ size and tissue homeostasis by regulating cell proliferation and apoptosis. The LATS-mediated negative feedback loop prevents excessive activation of the effectors YAP/TAZ, maintaining homeostasis of the Hippo pathway. YAP and TAZ are hyperactivated in various cancer cells which lead to tumor growth. Aberrantly increased O-GlcNAcylation has recently emerged as a cause of hyperactivation of YAP in cancer cells. However, the mechanism, which induces hyperactivation of TAZ and blocks LATS-mediated negative feedback, remains to be elucidated in cancer cells. This study found that in breast cancer cells, abnormally increased O-GlcNAcylation hyperactivates YAP/TAZ and inhibits LATS2, a direct negative regulator of YAP/TAZ. LATS2 is one of the newly identified O-GlcNAcylated components in the MST-LATS kinase cascade. Here, we found that O-GlcNAcylation at LATS2 Thr436 interrupted its interaction with the MOB1 adaptor protein, which connects MST to LATS2, leading to activation of YAP/TAZ by suppressing LATS2 kinase activity. LATS2 is a core component in the LATS-mediated negative feedback loop. Thus, this study suggests that LATS2 O-GlcNAcylation is deeply involved in tumor growth by playing a critical role in dysregulation of the Hippo pathway in cancer cells.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Proliferação de Células , Células HEK293 , Via de Sinalização Hippo , Homeostase , Humanos , FosforilaçãoRESUMO
BACKGROUND: Twin-to-twin transfusion syndrome (TTTS) is a serious complication of monochorionic twin pregnancies. It results from disproportionate blood supply to each fetus caused by abnormal vascular anastomosis within the placenta. Amniotic fluid (AF) is an indicator reflecting the various conditions of the fetus, and an imbalance in AF volume is essential for the antenatal diagnosis of TTTS by ultrasound. In this study, two different mass spectrometry quantitative approaches were performed to identify differentially expressed proteins (DEPs) within matched pairs of AF samples. METHODS: We characterized the AF proteome in pooled AF samples collected from donor and recipient twin pairs (n = 5 each) with TTTS by a global proteomics profiling approach and then preformed the statistical analysis to determine the DEPs between the two groups. Next, we carried out a targeted proteomic approach (multiple reaction monitoring) with DEPs to achieve high-confident TTTS-associated AF proteins. RESULTS: A total of 103 AF proteins that were significantly altered in their abundances between donor and recipient fetuses. The majority of upregulated proteins identified in the recipient twins (including carbonic anhydrase 1, fibrinogen alpha chain, aminopeptidase N, alpha-fetoprotein, fibrinogen gamma chain, and basement membrane-specific heparan sulfate proteoglycan core protein) have been associated with cardiac or dermatologic disease, which is often seen in recipient twins as a result of volume overload. In contrast, proteins significantly upregulated in AF collected from donor twins (including IgGFc-binding protein, apolipoprotein C-I, complement C1q subcomponent subunit B, apolipoprotein C-III, apolipoprotein A-II, decorin, alpha-2-macroglobulin, apolipoprotein A-I, and fibronectin) were those previously shown to be associated with inflammation, ischemic cardiovascular complications or renal disease. CONCLUSION: In this study, we identified proteomic biomarkers in AF collected from donor and recipient twins in pregnancies complicated by TTTS that appear to reflect underlying functional and pathophysiological challenges faced by each of the fetuses.
Assuntos
Líquido Amniótico/metabolismo , Transfusão Feto-Fetal/diagnóstico , Proteômica , Biomarcadores/metabolismo , Feminino , Humanos , Recém-Nascido , Placenta/metabolismo , Gravidez , Gravidez de Gêmeos , Diagnóstico Pré-Natal , ProteomaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Post-translational modifications, including O-GlcNAcylation, play fundamental roles in modulating cellular events, including transcription, signal transduction, and immune signaling. Several molecular targets of O-GlcNAcylation associated with pathogen-induced innate immune responses have been identified; however, the direct regulatory mechanisms linking O-GlcNAcylation with antiviral RIG-I-like receptor signaling are not fully understood. In this study, we found that cellular levels of O-GlcNAcylation decline in response to infection with Sendai virus. We identified a heavily O-GlcNAcylated serine-rich region between amino acids 249-257 of the mitochondrial antiviral signaling protein (MAVS); modification at this site disrupts MAVS aggregation and prevents MAVS-mediated activation and signaling. O-GlcNAcylation of the serine-rich region of MAVS also suppresses its interaction with TRAF3; this prevents IRF3 activation and production of interferon-ß. Taken together, these results suggest that O-GlcNAcylation of MAVS may be a master regulatory event that promotes host defense against RNA viruses.
Assuntos
Acetilglucosamina/imunologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Infecções por Respirovirus/imunologia , Vírus Sendai , Acilação , Linhagem Celular , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Mitocôndrias/imunologia , Transdução de SinaisRESUMO
The N-end rule pathway is a proteolytic system in which single N-terminal residues of proteins act as N-degrons. These degrons are recognized by N-recognins, facilitating substrate degradation via the ubiquitin (Ub) proteasome system (UPS) or autophagy. We have previously identified a set of N-recognins [UBR1, UBR2, UBR4 (also known as p600) and UBR5 (also known as EDD)] that bind N-degrons through their UBR boxes to promote proteolysis by the proteasome. Here, we show that the 570â kDa N-recognin UBR4 is associated with maturing endosomes through an interaction with Ca2+-bound calmodulin. The endosomal recruitment of UBR4 is essential for the biogenesis of early endosomes (EEs) and endosome-related processes, such as the trafficking of endocytosed protein cargos and degradation of extracellular cargos by endosomal hydrolases. In mouse embryos, UBR4 marks and plays a role in the endosome-lysosome pathway that mediates the heterophagic proteolysis of endocytosed maternal proteins into amino acids. By screening 9591 drugs through the DrugBank database, we identify picolinic acid as a putative ligand for UBR4 that inhibits the biogenesis of EEs. Our results suggest that UBR4 is an essential modulator in the endosome-lysosome system.This article has an associated First Person interview with the first author of the paper.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Endossomos/metabolismo , Cálcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Proteínas de Ligação a Calmodulina/genética , Proteínas do Citoesqueleto/genética , Endossomos/genética , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Biogênese de Organelas , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína LigasesRESUMO
The N-end rule pathway is a proteolytic system in which single N-terminal amino acids of proteins act as a class of degrons (N-degrons) that determine the half-lives of proteins. We have previously identified a family of mammals N-recognins (termed UBR1, UBR2, UBR4/p600, and UBR5/EDD) whose conserved UBR boxes bind N-degrons to facilitate substrate ubiquitination and proteasomal degradation via the ubiquitin-proteasome system (UPS). Amongst these N-recognins, UBR1 and UBR2 mediate ubiquitination and proteolysis of short-lived regulators and misfolded proteins. Here, we characterized the null phenotypes of UBR4-deficient mice in which the UBR box of UBR4 was deleted. We show that the mutant mice die around embryonic days 9.5-10.5 (E9.5-E10.5) associated with abnormalities in various developmental processes such as neurogenesis and cardiovascular development. These developmental defects are significantly attributed to the inability to maintain cell integrity and adhesion, which significantly correlates to the severity of null phenotypes. UBR4-loss induces the depletion of many, but not all, proteins from the plasma membrane, suggesting that UBR4 is involved in proteome-wide turnover of cell surface proteins. Indeed, UBR4 is associated with and required to generate the multivesicular body (MVB) which transiently store endocytosed cell surface proteins before their targeting to autophagosomes and subsequently lysosomes. Our results suggest that the N-recognin UBR4 plays a role in the homeostasis of cell surface proteins and, thus, cell adhesion and integrity.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Membrana Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Homeostase/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Neurogênese/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Autofagossomos/metabolismo , Proteínas de Ligação a Calmodulina/genética , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/genética , Endocitose/fisiologia , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisossomos/metabolismo , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Corpos Multivesiculares/metabolismo , Proteoma , Ubiquitina-Proteína Ligases/genéticaRESUMO
OBJECTIVE: Immune cells from patients with rheumatoid arthritis (RA) express more enolase-1 (ENO1) on their surface than those from healthy subjects, and they elicit an enhanced inflammatory response. This study is aimed to identify the ligands of ENO1 that could promote inflammatory loops in vitro and enhance the arthritis severity in vivo. METHODS: ENO1-binding proteins in RA synovial fluid were identified by mass spectromety, and affinity to ENO1 was evaluated by means of a ligand blotting and binding assay, surface plasmon resonance and confocal microscopy. Proinflammatory response by the interaction between ENO1 and apolipoprotein B (apoB) was tested in vitro and in vivo using peripheral blood mononuclear cells and a K/BxN serum transfer arthritis model and low-density lipoproteins receptor (LDLR) knockout mice. RESULTS: ApoB in the synovid fluid of patients with RA was identified as a specific ligand to ENO1 with a higher affinity than plasminogen, a known ENO1 ligand. ApoB binding to ENO1 on monocytes elicited the production of tumour necrosis factor-α, interleukins (IL)-1ß and IL-6 through both p38 mitogen-activated protein kinase and NF-κB pathways. In the K/BxN serum transfer arthritis model, administration of apoB increased the production of proinflammatory cytokines and exaggerated arthritis severity. The severity of K/BxN serum transfer arthritis in LDLR knockout mice was comparable with wild-type mice. CONCLUSIONS: A key component of atherogenic lipids, apoB, aggravated arthritis by potentiating the inflammatory response via its interaction with ENO1 expressed on the surface of immune cells. This suggests a novel mechanism by which lipid metabolism regulates chronic inflammation in RA.
Assuntos
Apolipoproteínas B/metabolismo , Artrite Reumatoide/metabolismo , Biomarcadores Tumorais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fosfopiruvato Hidratase/metabolismo , Líquido Sinovial/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Citocinas/biossíntese , Humanos , Inflamação , Leucócitos Mononucleares/metabolismo , NF-kappa B/metabolismo , Osteoartrite/metabolismo , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Recurrence and drug resistance of breast cancer are still the main reasons for breast cancer-associated deaths. Cancer stem cell (CSC) model has been proposed as a hypothesis for the lethality of breast cancer. Molecular mechanisms underlying CSC maintenance are still unclear. In this study, we generated mammospheres derived from breast cancer MDA-MB231 cells and MCF7 cells to enrich CSCs and performed DNA microarray analysis. We found that the expression of carboxy terminus of HSP70-interacting protein (CHIP) E3 ubiquitin ligase was significantly downregulated in breast CSCs. CHIP depletion increased mammosphere formation, whereas CHIP overexpression reversed this effect. We identified interactomes by mass spectrometry and detected CHIP directly interacted with OCT4, a stemness factor. CHIP overexpression decreased OCT4 stability through proteasomal degradation. CHIP induced OCT4 ubiquitination, whereas H260Q, a catalytic CHIP mutant, did not. Interestingly, we determined that OCT4 was ubiquitinated at lysine 284, and CHIP overexpression did not degrade K284R mutant OCT4. CHIP overexpression decreased the proliferation and side population of breast cancer cells, but these were not occurred in K284R mutant OCT4 overexpressed cells. Only 1000 cells showing CHIP depletion or OCT4 overexpression sufficiently generated breast tumors and lung metastases in xenografted mice. Ubiquitination-defective mutant of OCT4(K284R) overexpressed cells drastically generated tumor burdens in mice. Patients with breast cancer who showed low CHIP expression had poor survival probability. Taken together, we suggest that CHIP-induced OCT4 ubiquitination is important in breast CSCs. Regulation of CHIP expression and OCT4 protein stability is a considerable approach for breast cancer therapy.
Assuntos
Fator 3 de Transcrição de Octâmero/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinogênese , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Células MCF-7 , Camundongos , Camundongos Nus , Mutagênese Sítio-Dirigida , Fator 3 de Transcrição de Octâmero/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transplante Heterólogo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
The proto-oncogene tyrosine kinase ROS1 plays a key role in carcinogenesis through gene rearrangement to form a fusion protein with other genes, in which the C-terminal intracellular region of ROS1 participates. The possibility of wild type ROS1 overexpression through epigenetic regulation has been proposed. Here, we generated an antibody, 3B20, reactive to the N-terminal region of ROS1 to use it for the detection of wild type ROS1 in cancerous tissues. Using immunoblot and immunoprecipitation analyses, we found that 3B20 also reacted with heat shock proteins (Hsp)70s. Using homology searching, ROS1 and Hsp70s were found to share an identical amino acid sequence: DLGT. Using alanine mutagenesis of ROS1, the epitope was found to harbor this sequence. To modify the idiotope with the aim of selecting more specific antibodies, we introduced random mutations into the heavy chain complementarity-determining region 3 and successfully generated an antibody clone, 3B20-G1K, with a point mutation that only reacted with ROS1 in enzyme-linked immunosorbent assays, and in immunoblot and immunoprecipitation analysis. In immunohistochemical analysis using 3B20-G1K, ROS1 was found to be absent in normal lung tissues and was overexpressed in a case of lung adenocarcinoma.
Assuntos
Adenocarcinoma/imunologia , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Antineoplásicos/imunologia , Proteínas de Arabidopsis/genética , Neoplasias Pulmonares/imunologia , Proteínas Nucleares/genética , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Anticorpos Monoclonais/administração & dosagem , Antineoplásicos/administração & dosagem , Proteínas de Arabidopsis/imunologia , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Desenho de Fármacos , Sinergismo Farmacológico , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Mutagênese Sítio-Dirigida/métodos , Proteínas Nucleares/imunologia , Mutação Puntual/genética , Proto-Oncogene Mas , Proto-Oncogenes , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
Nucleocytoplasmic O-GlcNAc transferase (OGT) attaches a single GlcNAc to hydroxyl groups of serine and threonine residues. Although the cellular localisation of OGT is important to regulate a variety of cellular processes, the molecular mechanisms regulating the nuclear localisation of OGT is unclear. Here, we characterised three amino acids (DFP; residues 451-453) as the nuclear localisation signal of OGT and demonstrated that this motif mediated the nuclear import of non-diffusible ß-galactosidase. OGT bound the importin α5 protein, and this association was abolished when the DFP motif of OGT was mutated or deleted. We also revealed that O-GlcNAcylation of Ser389, which resides in the tetratricopeptide repeats, plays an important role in the nuclear localisation of OGT. Our findings may explain how OGT, which possesses a NLS, exists in the nucleus and cytosol simultaneously.
Assuntos
Núcleo Celular/enzimologia , N-Acetilglucosaminiltransferases/metabolismo , Sinais de Localização Nuclear/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Núcleo Celular/genética , Células HEK293 , Células HeLa , Humanos , N-Acetilglucosaminiltransferases/genética , Sinais de Localização Nuclear/genéticaRESUMO
Defective deep placentation, involving abnormal transformation of the spiral arteries in the junctional zone of the myometrium, is known to cause significant obstetric complications, such as preeclampsia (PE), fetal growth restriction, and placental infarction leading to fetal death. Serological biomarkers to predict and diagnose PE would help antenatal care and reduce obstetric complications. To discover candidate PE biomarkers, we first performed global proteomic profiling of three pairs of plasma samples obtained from pregnant women in the early second trimester, who subsequently developed PE, and controls to identify candidate proteins that were abundant in the patients. We further evaluated the changes in the expression of PE-representing proteins in stored plasma samples of a cohort that subsequently developed PE and their matched controls by MRM-MS analysis. We identified that both complement C1s subcomponent (C1S) and protein AMBP were elevated in the plasma samples of the PE cohort before the manifestation of clinical disease. We propose that these proteins may be involved in the remodeling process of the spiral arteries even before PE manifestation. These proteins can serve as potential plasma biomarkers to predict the pregnant women having an increased risk of developing PE.
Assuntos
Expressão Gênica , Pré-Eclâmpsia/sangue , Sequência de Aminoácidos , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Regulação da Expressão Gênica , Humanos , Gravidez , ProteômicaRESUMO
Glycosylation with O-linked ß-N-acetylglucosamine (O-GlcNAc) is one of the protein glycosylations affecting various intracellular events. However, the role of O-GlcNAcylation in neurodegenerative diseases such as Alzheimer's disease (AD) is poorly understood. Mitochondrial adenosine 5'-triphosphate (ATP) synthase is a multiprotein complex that synthesizes ATP from ADP and Pi. Here, we found that ATP synthase subunit α (ATP5A) was O-GlcNAcylated at Thr432 and ATP5A O-GlcNAcylation was decreased in the brains of AD patients and transgenic mouse model, as well as Aß-treated cells. Indeed, Aß bound to ATP synthase directly and reduced the O-GlcNAcylation of ATP5A by inhibition of direct interaction between ATP5A and mitochondrial O-GlcNAc transferase, resulting in decreased ATP production and ATPase activity. Furthermore, treatment of O-GlcNAcase inhibitor rescued the Aß-induced impairment in ATP production and ATPase activity. These results indicate that Aß-mediated reduction of ATP synthase activity in AD pathology results from direct binding between Aß and ATP synthase and inhibition of O-GlcNAcylation of Thr432 residue on ATP5A.
Assuntos
Doença de Alzheimer/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Fatores Acopladores da Fosforilação Oxidativa/metabolismo , Acetilglucosamina/metabolismo , Trifosfato de Adenosina/metabolismo , Doença de Alzheimer/enzimologia , Doença de Alzheimer/genética , Animais , Células CHO , Cricetulus , Modelos Animais de Doenças , Glicosilação , Células HeLa , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , Fatores Acopladores da Fosforilação Oxidativa/genética , Processamento de Proteína Pós-Traducional , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
Splice variant forms of neuronal nitric oxide synthase (nNOS or NOS1), nNOSα and nNOSµ, are well established to be functionally expressed in discrete compartments in cardiomyocytes (e.g. sarcoplasmic reticulum, SR, caveolae in plasma membrane or mitochondria). So far, whether nNOS is expressed in myofilament fraction of cardiomyocytes and the splice variant form of nNOS are unknown. Immunoblotting results using two nNOS specific antibodies (BD Transduction Laboratories aa 1095-1289 and Santa Cruz Biotechnology aa 2-300) clearly demonstrated that nNOS was abundantly expressed in myofilament-enriched fraction of cardiomyocytes. Whilst the molecular weight of nNOS in membrane/cytosol fractions was â¼165 kDa, nNOS in myofilament was below 140 kDa, suggesting that the predominant splice variant of nNOS in myofilament is nNOSß. RT-PCR results confirmed the expressions of both nNOSα and nNOSß mRNAs in rat cardiomyocytes. Similarly, immunoprecipitation experiments using myofilament lysates of cardiomyocytes identified nNOS with low molecular weight (M.W. â¼140 kDa), confirming nNOSß. Intriguingly, all three splice variants of nNOS were undetectable in the lysates of cardiomyocytes (including myofilament fractions) from nNOS-/- mice (which lacks nNOSα/µ). Furthermore, nNOSß expression in myofilament of cardiomyocytes was not different in hypertensive rats compared to the level expressed in sham. iTRAQ-based quantitative proteomics analysis revealed that nNOS regulates phosphorylations of â¼20 proteins in cardiac myofilaments. Collectively, we provide direct evidence that different splice variants of nNOS are expressed in myofilament and membrane/cytosol fractions of cardiomyocytes. Discrete expressions of various splice variants in different compartments of cardiomyocytes suggest diverse roles nNOS play in healthy and diseased heart.
RESUMO
Aberrant glutathione or Ca(2+) homeostasis due to oxidative stress is associated with the pathogenesis of neurodegenerative disorders. The Ca(2+)-permeable transient receptor potential cation (TRPC) channel is predominantly expressed in the brain, which is sensitive to oxidative stress. However, the role of the TRPC channel in neurodegeneration is not known. Here, we report a mechanism of TRPC5 activation by oxidants and the effect of glutathionylated TRPC5 on striatal neurons in Huntington's disease. Intracellular oxidized glutathione leads to TRPC5 activation via TRPC5 S-glutathionylation at Cys176/Cys178 residues. The oxidized glutathione-activated TRPC5-like current results in a sustained increase in cytosolic Ca(2+), activated calmodulin-dependent protein kinase and the calpain-caspase pathway, ultimately inducing striatal neuronal cell death. We observed an abnormal glutathione pool indicative of an oxidized state in the striatum of Huntington's disease transgenic (YAC128) mice. Increased levels of endogenous TRPC5 S-glutathionylation were observed in the striatum in both transgenic mice and patients with Huntington's disease. Both knockdown and inhibition of TRPC5 significantly attenuated oxidation-induced striatal neuronal cell death. Moreover, a TRPC5 blocker improved rearing behaviour in Huntington's disease transgenic mice and motor behavioural symptoms in littermate control mice by increasing striatal neuron survival. Notably, low levels of TRPC1 increased the formation of TRPC5 homotetramer, a highly Ca(2+)-permeable channel, and stimulated Ca(2+)-dependent apoptosis in Huntington's disease cells (STHdh(Q111/111)). Taken together, these novel findings indicate that increased TRPC5 S-glutathionylation by oxidative stress and decreased TRPC1 expression contribute to neuronal damage in the striatum and may underlie neurodegeneration in Huntington's disease.
Assuntos
Corpo Estriado/patologia , Glutationa/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Doença de Huntington/patologia , Neurônios/metabolismo , Canais de Cátion TRPC/metabolismo , Análise de Variância , Animais , Cálcio/metabolismo , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Proteína Huntingtina , Camundongos , Camundongos Transgênicos , Mutação/genética , Proteínas do Tecido Nervoso/genética , RNA Interferente Pequeno/metabolismo , Canais de Cátion TRPC/genética , TransfecçãoRESUMO
For cells to exit from pluripotency and commit to a lineage, the circuitry of a core transcription factor (CTF) network must be extinguished in an orderly manner through epigenetic modifications. However, how this choreographed epigenetic remodeling at active embryonic stem cell (ESC) genes occurs during differentiation is poorly understood. In this study, we demonstrate that C-terminal binding protein 2 (Ctbp2) regulates nucleosome remodeling and deacetylation (NuRD)-mediated deacetylation of H3K27 and facilitates recruitment of polycomb repressive complex 2 (PRC2)-mediated H3K27me3 in active ESC genes for exit from pluripotency during differentiation. By genomewide analysis, we found that Ctbp2 resides in active ESC genes and co-occupies regions with ESC CTFs in undifferentiated ESCs. Furthermore, ablation of Ctbp2 effects inappropriate gene silencing in ESCs by sustaining high levels of H3K27ac and impeding H3K27me3 in active ESC genes, thereby sustaining ESC maintenance during differentiation. Thus, Ctbp2 preoccupies regions in active genes with the NuRD complex in undifferentiated ESCs that are directed toward H3K27me3 by PRC2 to induce stable silencing, which is pivotal for natural lineage commitment.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Epigênese Genética/fisiologia , Histonas/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Repressoras/metabolismo , Oxirredutases do Álcool , Animais , Linhagem Celular , Montagem e Desmontagem da Cromatina/fisiologia , Proteínas Correpressoras , Proteínas de Ligação a DNA/genética , Histonas/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Nucleossomos/genética , Nucleossomos/metabolismo , Fosfoproteínas/genética , Proteínas Repressoras/genéticaRESUMO
Indoleamine 2,3-dioxygenases (IDOs) are tryptophan-catabolizing enzymes with immunomodulatory functions. However, the biological role of IDO2 and its relationship with IDO1 are unknown. To assess the relationship between IDO2 and IDO1, we investigated the effects of co-expression of human (h) IDO2 on hIDO1 activity. Cells co-expressing hIDO1 and hIDO2 showed reduced tryptophan metabolic activity compared with those expressing hIDO1 only. In a proteomic analysis, hIDO1-expressing cells exhibited enhanced expression of proteins related to the cell cycle and amino acid metabolism, and decreased expression of proteins related to cell survival. However, cells co-expressing hIDO1 and hIDO2 showed enhanced expression of negative regulators of cell apoptosis compared with those expressing hIDO1 only. Co-expression of hIDO1 and hIDO2 rescued the cell death induced by tryptophan-depletion through hIDO1 activity. Cells expressing only hIDO2 exhibited no marked differences in proteome profiles or cell growth compared with mock-transfectants. Cellular tryptophan metabolic activity and cell death were restored by co-expressing the hIDO2 mutant substituting the histidine 360 residue for alanine. These results demonstrate that hIDO2 plays a novel role as a negative regulator of hIDO1 by competing for heme-binding with hIDO1, and provide information useful for development of therapeutic strategies to control cancer and immunological disorders that target IDO molecules.
Assuntos
Heme/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Triptofano/metabolismo , Proliferação de Células , Sobrevivência Celular , Expressão Gênica , Células HEK293 , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Ligação Proteica , Regulação para CimaRESUMO
Current serum biomarkers for rheumatoid arthritis (RA) are not highly sensitive or specific to changes of disease activities. Thus, other complementary biomarkers have been needed to improve assessment of RA activities. In many diseases, urine has been studied as a window to provide complementary information to serum measures. Here, we conducted quantitative urinary proteome profiling using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and identified 134 differentially expressed proteins (DEPs) between RA and osteoarthritis (OA) urine samples. By integrating the DEPs with gene expression profiles in joints and mononuclear cells, we initially selected 12 biomarker candidates related to joint pathology and then tested their altered expression in independent RA and OA samples using enzyme-linked immunosorbent assay. Of the initial candidates, we selected four DEPs as final candidates that were abundant in RA patients and consistent with those observed in LC-MS/MS analysis. Among them, we further focused on urinary soluble CD14 (sCD14) and examined its diagnostic value and association with disease activity. Urinary sCD14 had a diagnostic value comparable to conventional serum measures and an even higher predictive power for disease activity when combined with serum C-reactive protein. Thus, our urinary proteome provides a diagnostic window complementary to current serum parameters for the disease activity of RA.
Assuntos
Artrite Reumatoide/urina , Receptores de Lipopolissacarídeos/urina , Proteinúria/urina , Proteômica/métodos , Artrite Reumatoide/etiologia , Biomarcadores/urina , Proteína C-Reativa/análise , Estudos de Casos e Controles , Cromatografia Líquida/métodos , Estudos de Coortes , Humanos , Lúpus Eritematoso Sistêmico/urina , Osteoartrite/urina , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Líquido Sinovial/fisiologia , Espectrometria de Massas em Tandem/métodosRESUMO
Autoantibodies can facilitate diagnostic and therapeutic means for type 1 diabetes (T1DM). We profiled autoantibodies from serum samples of 16 T1DM patients, 16 type 2 diabetic (T2DM) patients, and 27 healthy control subjects with normal glucose tolerance (NGT) by using protein microarrays containing 9,480 proteins. Two novel autoantibodies, anti-EEF1A1 and anti-UBE2L3, were selected from microarrays followed by immunofluorescence staining of pancreas. We then tested the validity of the candidates by ELISA in two independent test cohorts: 1) 95 adults with T1DM, 49 with T2DM, 11 with latent autoimmune diabetes in adults (LADA), 20 with Graves disease, and 66 with NGT and 2) 33 children with T1DM and 34 healthy children. Concentrations of these autoantibodies were significantly higher in T1DM patients than in NGT and T2DM subjects (P < 0.01), which was also confirmed in the test cohort of children (P < 0.05). Prevalence of anti-EEF1A1 and anti-UBE2L3 antibodies was 29.5% and 35.8% in T1DM, respectively. Of note, 40.9% of T1DM patients who lack anti-GAD antibodies (GADA) had anti-EEF1A1 and/or anti-UBE2L3 antibodies. These were also detected in patients with fulminant T1DM but not LADA. Our approach identified autoantibodies that can provide a new dimension of information indicative of T1DM independent of GADA and new insights into diagnosis and classification of T1DM.